EMBO Reports Research Articles

Abstract 4376: The pseudokinase PTK7 is a negative regulator of EPHA2 in colorectal cancer

Abstract Colorectal cancer (CRC) remains a major public health issue. Identification of biomarkers predicting high risk of recurrence and discovery of novel targets for therapy are urgently needed1. The cell surface receptor PTK7 is a poorly described evolutionary conserved member of the receptor tyrosine kinase superfamily that was first identified in human colon carcinoma and melanoma2-3. Accumulated data highlighted the implication of PTK7 in diverse cancer-related signaling pathways4-6 and, despite the lack of kinase activity, anti-PTK7 inhibitors are currently in development at preclinical and clinical stages7-9. The dual aim of this project is to increase the basic knowledge about PTK7 and to evaluate its role in CRC in order to propose in the future novel therapeutics directed against PTK7. With the aim to discover novel PTK7 associated pathways, we used a proximity biotinylation strategy coupled to mass spectrometry in CRC cells. Among the partners identified, we found that the pseudokinase PTK7 interacts with the active receptor tyrosine kinase EphrinA2 (EPHA2). Similarly to PTK7, EPHA2 expression correlates with poor prognosis in CRC10. In this work, we also demonstrate that PTK7 acts as a negative regulator of EPHA2 activation through the promotion of distinct oligomeric protein complexes and the modulation of EPHA2 phosphorylation. We also shed in light on the role of PTK7 in EPHA2 K63-linked ubiquitination and its subsequent endosomal sorting and lysosomal degradation. References 1 Punt CJ et al, Nat. Rev. Clin. Oncol., 2016; 2 Lhoumeau AC et al, Cell Cycle, 2011; 3 Lhoumeau AC et al, PLoS One, 2015; 4 Puppo F et al, EMBO reports, 2011; 5 Martinez S et al, J. Biol. Chem., 2015; 6 Daulat AM and Borg JP, Trends in Cancer, 2017; 7 Damelin M et al, Sci. Transl. Med., 2017; 8 Jie Y et al, Front. Immunol., 2021; 9 Maitland ML et al, Clin. Cancer. Res., 2021; 10 Dunne P et al, Clin. Cancer. Res., 2016; 11 Cioce M and Fazio VM, Cancers, 2021. Citation Format: Jean-Paul Borg, Charlotte Dessaux, Luc Camoin, Stéphane Audebert, Emilie Baudelet, Avais Daulat. The pseudokinase PTK7 is a negative regulator of EPHA2 in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4376.

Ruthenium red: Blocker or antagonist of TRPV channels?

Ruthenium red (RR) is a widely used inhibitor of Transient Receptor Potential (TRP) cation channels and other types of ion channels. Although RR has been generally accepted to inhibit TRP channels by physically blocking the ion permeation pathway, recent structural evidence suggests that it might also function as an antagonist, inducing conformational changes in the channel upon binding that result in closure of the pore. In a recent manuscript published in EMBO Reports, Ruth A. Pumroy and collaborators solve structures of TRPV2 and TRPV5 channels in the presence and absence of activators and RR. The data sheds light on the mechanism of inhibition by RR, while also opening new questions for further investigation.

Natural probiotics improve heritable sterility.

Disrupting the small RNA pathway and chromatin-modifying enzymes in C. elegans often leads to a mortal germline (Mrt) phenotype, characterized by progressive sterility observed over multiple generations at elevated temperature. This phenotype arises from the inheritance of aberrant epigenetic memory across generations. In this issue of EMBO Reports, Frézal and colleagues reported that, while in standard laboratory environment C. elegans wild isolates exhibit the Mrt phenotype, sterility does not occur when the worms are exposed to naturally associated bacteria and microsporidia. Excitingly, diet-induced epigenetic memory may persist for multiple generations. This suggests intriguing diet-gene interactions in modulating nongenetic inheritance, potentially shaping the evolutionary trajectory of the animals.

Hematopoiesis: a BETter understanding.

Epigenetic modifications are known to be crucial for hematopoietic stem cell (HSC) differentiation, with the BET family member BRD4 playing a vital role in this as an epigenetic reader. In this issue of EMBO reports, Yang etal (2023) demonstrate that the absence of BRD4 leads to senescence in HSCs and hematopoietic progenitor cells (HPCs), affecting the expression of crucial genes involved in myeloid and erythroid development. These data suggest that BRD4 has a protective role in preserving histone tails, thereby sustaining normal HSC/HPC functions.

TECPR1 helps bridge the CASM during lysosome damage.

Maintaining the integrity of the endolysosomal system is of great importance forcellular homeostasis. Recent work published in The EMBO Journal and EMBO Reports reveals a novel role for the protein TECPR1 as a sensor for stressed membranes and regulator of lysosomal membrane repair.

Spns2/S1P: it takes two to tango with inflammation and metabolic rewiring during sepsis.

Sepsis is the result of a dysregulated host response to an infection and causes high morbidity and mortality at the intensive care units worldwide. Despite intensive research, the current management of sepsis is supportive rather than curative. Therefore, new therapeutic interventions for sepsis and septic shock patients are urgently needed. In this issue of EMBO Reports, Fang etal have used rat sepsis models to show that macrophage-expressed SPNS2, a major transporter of S1P, is a crucial mediator of metabolic reprogramming of macrophages during sepsis which regulates inflammation via the lactate-ROS axis.

The potential role of Fibrillin 2 in equine placental and fetal development

Energy adaption in pregnant dams is a vital process to appropriately supply the energy requirements to the fetus through the placenta (Yu et al. EMBO Reports (2020); Fowden et al. Equine vet. J. (1984) 16 (4), 239-246). The placenta is a transient organ with a crucial role in the survival and development of the fetus by providing nutrients, removing waste, and secreting hormones and growth factors. To understand the dynamics of the genes responsible for placental function, we recently investigated the equine placental DNA methylome. Chorioallantois (CA) samples were collected from healthy pregnant mares at different gestational time points (4, 6, and 10 months; n=3 for each time point). DNA was extracted and RRBS (Reduced Representation Bisulfate Sequencing) was performed. We then combined the methylome data with available transcriptomic profiles of the same samples to better detail the placental gene expression dynamics. Based on the reverse relationship between methylation and gene expression, we identified 18 genes with higher expression and lower methylation at 4 months compared to the other time points, including Fibrillin 2 (FBN2). Given the known high expression pattern of FBN2 in the human placenta, especially during early pregnancy in cytotrophoblasts, we decided to further detail its potential role in equine placental development. FBN2 encodes a peptide hormone called placensin that is secreted by trophoblasts and increases cAMP-PKA signaling. In humans, placensin increases gluconeogenesis and glucoserelease which stimulate cAMP release and PKA activity in maternal circulation and therefore, acts as a placental gluconeogenic hormone that increases in serum as pregnancy advances and the insulin resistance level increases. The changes in the glucose level and insulin resistance have also been documented in horses, however, the underlying mechanism has not yet been identified (Fowden et al. Equine vet. J. (1984) 16 (4), 239-246; George et al. Am J Vet Res 2011; 72:666–674). Our preliminary data suggests a possible involvement of FBN2 in gluconeogenesis in mares given its expression pattern. Since maternal glucose is the main source of energy for the fetus, any alteration in maternal glucose and insulin could have an effect on fetal development, which has been described in other species such as sheep (Yu et al. EMBO Reports (2020)). Further detailing the expression dynamics of FBN2 in placenta and placensin in maternal blood could aid elucidation of the mechanisms for glucose homeostasis in pregnant mares. Then, such analyses may be expanded to the additional genes showing similar expression dynamics to further highlight their potential role in placental development. This project was supported by the Center for Equine Health with funds provided by the State of California satellite wagering fund and contributions by private donors.

The fungal grid: Fungal communication via electrical signals has inspired the hypothesis of a Wood Wide Web of plants and fungi: Fungal communication via electrical signals has inspired the hypothesis of a Wood Wide Web of plants and fungi.

The observation that soil-dwelling fungi seem to exchange information via electrical impulses has raised new interest about their interactions with plants and their ecological significance.

Mcrs1 regulates G2/M transition and spindle assembly during mouse oocyte meiosis.

Microspherule protein 1 (Mcrs1) is a component of the nonspecific lethal (NSL) complex and the chromatin remodeling INO80 complex, which participates in transcriptional regulation during mitosis. Here, we investigate the roles of Mcrs1 during female meiosis in mice. We demonstrate that Mcrs1 is a novel regulator of the meiotic G2/M transition and spindle assembly in mouse oocytes. Mcrs1 is present in the nucleus and associates with spindle poles and chromosomes of oocytes during meiosis I. Depletion of Mcrs1 alters HDAC2-mediated H4K16ac, H3K4me2, and H3K9me2 levels in nonsurrounded nucleolus (NSN)-type oocytes, and reduces CDK1 activity and cyclin B1 accumulation, leading to G2/M transition delay. Furthermore, Mcrs1 depletion results in abnormal spindle assembly due to reduced Aurora kinase (Aurka and Aurkc) and Kif2A activities, suggesting that Mcrs1 also plays a transcription-independent role in regulation of metaphase I oocytes. Taken together, our results demonstrate that the transcription factor Mcrs1 has important roles in cell cycle regulation and spindle assembly in mouse oocyte meiosis.

Goliath induces inflammation in obese mice by linking fatty acid β-oxidation to glycolysis.

Obesity is associated with metabolic disorders and chronic inflammation. However, the obesity-associated metabolic contribution to inflammatory induction remains elusive. Here, we show that, compared with lean mice, CD4+ T cells from obese mice exhibit elevated basal levels of fatty acid β-oxidation (FAO), which promote T cell glycolysis and thus hyperactivation, leading to enhanced induction of inflammation. Mechanistically, the FAO rate-limiting enzyme carnitine palmitoyltransferase 1a (Cpt1a) stabilizes the mitochondrial E3 ubiquitin ligase Goliath, which mediates deubiquitination of calcineurin and thus enhances activation of NF-AT signaling, thereby promoting glycolysis and hyperactivation of CD4+ T cells in obesity. We also report the specific GOLIATH inhibitor DC-Gonib32, which blocks this FAO-glycolysis metabolic axis in CD4+ T cells of obese mice and reduces the induction of inflammation. Overall, these findings establish a role of a Goliath-bridged FAO-glycolysis axis in mediating CD4+ T cell hyperactivation and thus inflammation in obese mice.

SPTing across condensates: SEC-mediated translocation of SPT complex from pausing condensates to elongation condensates.

Several independent studies in the last few years have suggested that phase separation and biomolecular condensation play a critical role in regulating different transcription steps from initiation to pausing and elongation. However, how components of the transcription machinery translocate among different types of condensates during transcription remains poorly understood. Guo etal have now identified a potential mechanism underlying translocation of the DSIF complex from pausing to elongation condensates during promoter pause release, as reported in this issue of EMBO reports.

Selection and induction? Using ITGB4 to track the role of selection and constriction-induced changes in phenotype and 3D genome structure shifts after constricted migration

Metastatic cancer cells squeeze through small pores, often smaller than their nucleus, in order to reach a secondary site of proliferation. This constriction exerts forces on the nucleus, leading to deformations. Our previous work showed that A375 melanoma cells that have passed through 10 rounds of constricted migration exhibit stable differences in migratory phenotype, gene expression, and 3D genome structure (Golloshi et al. EMBO Reports, 2022). We are now investigating what combination of selection and induction forces lead to this stable difference. We have identified a cell surface protein, Integrin-β4 (ITGB4), as a marker whose RNA and protein expression level closely associates with cellular constricted migration proficiency over the course of sequential rounds of constriction. Sorting cells for expression of ITGB4 revealed a subpopulation of cells that, before constriction, already possess some of the phenotypic and 3D genome structure characteristics of proficient migrators. We next carefully tracked ITGB4 protein levels over a timecourse of sequential constricted migration, compared to unconstricted migration, in both the initial A375 population and a clonal population. We found evidence for both selection of ITGB4 expressing cells and also induction of ITGB4 expression after multiple constrictions. Intriguingly, this careful timecourse revealed that it consistently takes 3-4 rounds of constriction before a dramatic and stable shift in cell migratory phenotype. To investigate the molecular underpinnings of this lag, we collected nucleus images, Hi-C chromosome contact data, and gene expression data after 1 and 4 rounds of constriction. We propose that stable phenotypic differences after sequential constricted migration are the product of selection from a continuously regenerated heterogeneity combined with changes induced by the cumulative impact of multiple rounds of cell and nucleus constriction.

Thrombopoietin-independent Megakaryocyte Differentiation of Hematopoietic Progenitor Cells from Patients with Myeloproliferative Neoplasms.

Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient-derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34+ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41+ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders. This protocol was validated in: EMBO Rep (2022), DOI: 10.15252/embr.202152904 Graphical abstract Schematic representation of the isolation of CD34+ progenitor cells and subsequent TPO-independent megakaryocyte differentiation.

Sphingosine-1-phosphate controls endothelial sphingolipid homeostasis via ORMDL.

Disruption of sphingolipid homeostasis and signaling has been implicated in diabetes, cancer, cardiometabolic, and neurodegenerative disorders. Yet, mechanisms governing cellular sensing and regulation of sphingolipid homeostasis remain largely unknown. In yeast, serine palmitoyltransferase, catalyzing the first and rate-limiting step of sphingolipid de novo biosynthesis, is negatively regulated by Orm1 and 2. Lowering sphingolipids triggers Orms phosphorylation, upregulation of serine palmitoyltransferase activity and sphingolipid de novo biosynthesis. However, mammalian orthologs ORMDLs lack the N-terminus hosting the phosphosites. Thus, which sphingolipid(s) are sensed by the cells, and mechanisms of homeostasis remain largely unknown. Here, we identify sphingosine-1-phosphate (S1P) as key sphingolipid sensed by cells via S1PRs to maintain homeostasis. The increase in S1P-S1PR signaling stabilizes ORMDLs, restraining SPT activity. Mechanistically, the hydroxylation of ORMDLs at Pro137 allows a constitutive degradation of ORMDLs via ubiquitin-proteasome pathway, preserving SPT activity. Disrupting S1PR/ORMDL axis results in ceramide accrual, mitochondrial dysfunction, impaired signal transduction, all underlying endothelial dysfunction, early event in the onset of cardio- and cerebrovascular diseases. Our discovery may provide the molecular basis for therapeutic intervention restoring sphingolipid homeostasis.

Time required for commitment to T cell proliferation depends on TCR affinity and cytokine response.

T cell activation and effector functions are determined by the affinity of the interaction between T cell receptor (TCR) and its antigenic peptide MHC (pMHC) ligand. A better understanding of the quantitative aspects of TCR-pMHC affinity-dependent T cell activation is critical for the development of new immunotherapeutic strategies. However, the role of TCR-pMHC affinity in regulating the kinetics of CD8+ T cell commitment to proliferation and differentiation is unknown. Here, we show that the stronger the TCR-pMHC affinity, the shorter the time of T cell-APC co-culture required to commit CD8+ T cells to proliferation. The time threshold for T cell cytokine production is much lower than that for cell proliferation. There is a strong correlation between affinity-dependent differences in AKT phosphorylation and T cell proliferation. The cytokine IL-15 increases the poor proliferation of T cells stimulated with low affinity pMHC, suggesting that pro-inflammatory cytokines can override the affinity-dependent features of T cell proliferation.

Equally potent: Nlrp3 mutation in macrophages or neutrophils is sufficient to drive autoinflammation.

Gain-of-function mutation in NLRP3 is associated with a spectrum of autoinflammatory disorders including familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and neonatal onset multisystem inflammatory disease, collectively known as cryopyrin-associated periodic syndrome (CAPS). However, the cell types mediating the pathogenesis of CAPS are not completely understood. Two studies in EMBO Reports now demonstrate that gain-of-function Nlrp3 mutation in either macrophages or neutrophils alone is sufficient to trigger systemic autoinflammation and lethality in mice.

Interleukin-38 promotes skin tumorigenesis in an IL-1Rrp2-dependent manner.

Interleukin-38 (IL-38) is strongly associated with chronic inflammatory diseases; however, its role in tumorigenesis is poorly understood. We demonstrated that expression of IL-38, which exhibits high expression in the skin, is downregulated in human cutaneous squamous cell carcinoma and 7,12-dimethylbenzanthracene/12-O-tetradecanoyl phorbol-13-acetate-induced mouse skin tumorigenesis. IL-38 keratinocyte-specific knockout mice displayed suppressed skin tumor formation and malignant progression. Keratinocyte-specific deletion of IL-38 was associated with reduced expression of inflammatory cytokines, leading to reduced myeloid cell infiltration into the local tumor microenvironment. IL-38 is dispensable for epidermal mutagenesis, but IL-38 keratinocyte-specific deletion reduces proliferative gene expression along with epidermal cell proliferation and hyperplasia. Mechanistically, we first demonstrated that IL-38 activates the c-Jun N-terminal kinase (JNK)/activator protein 1 signal transduction pathway to promote the expression of cancer-related inflammatory cytokines and proliferation and migration of tumor cells in an IL-1 receptor-related protein 2 (IL-1Rrp2)-dependent manner. Our findings highlight the role of IL-38 in the regulation of epidermal cell hyperplasia and pro-tumorigenic microenvironment through IL-1Rrp2/JNK and suggest IL-38/IL-1Rrp2 as a preventive and potential therapeutic target in skin cancer.

The international of wisdom.

Russia's invasion of Ukraine is the continuation of Soviet communism's aggressive expansion. But as under communism, freedom of thought will prevail.

KLF10 promotes nonalcoholic steatohepatitis progression through transcriptional activation of zDHHC7.

Nonalcoholic steatohepatitis (NASH), characterized by hepatic steatosis, inflammation, and liver injury, has become a leading cause of end-stage liver diseases and liver transplantation. Krüppel-like factors 10 (KLF10) is a Cys2/His2 zinc finger transcription factor that regulates cell growth, apoptosis, and differentiation. However, whether it plays a role in the development and progression of NASH remains poorly understood. In the present study, we found that KLF10 expression was selectively upregulated in the mouse models and human patients with NASH, compared with simple steatosis (NAFL). Gain- and loss-of function studies demonstrated that hepatocyte-specific overexpression of KLF10 aggravated, whereas its depletion alleviated diet-induced NASH pathogenesis in mice. Mechanistically, transcriptomic analysis and subsequent functional experiments showed that KLF10 promotes hepatic lipid accumulation and inflammation through the palmitoylation and plasma membrane localization of fatty acid translocase CD36 via transcriptionally activation of zDHHC7. Indeed, both expression of zDHHC7 and palmitoylation of CD36 are required for the pathogenic roles of KLF10 in NASH development. Thus, our results identify an important role for KLF10 in NAFL-to-NASH progression through zDHHC7-mediated CD36 palmitoylation.

Disruption of ATG9A‐dependent basal autophagy causes an accumulation of ubiquitin‐rich condensates which act as a platform for inflammatory signaling

Excessive inflammation underlies many human diseases, such as neurodegeneration, autoimmune disorders, cancer, and COVID‐19. Thus, cellular mechanisms that control inflammation are of high therapeutic interest. Autophagy has been implicated in the suppression of inflammation, yet mechanistic links between autophagy and inflammation are not completely understood. Previous work demonstrated that loss of ATG9A, but not ATG5 or ATG7, increased inflammatory signaling through the STING‐IRF3 cascade, suggesting that perhaps an autophagy‐independent function of ATG9A regulates inflammation.1 Here, we show that loss of the essential basal autophagy regulators ATG9A and ATG101 sensitizes cells to dsDNA‐induced IRF3 activation. Importantly, this effect was not observed with the loss of ATG5 or ATG7, suggesting that the canonical LC3‐lipidation autophagy machinery is not required for suppression of IRF3 and inflammatory signaling.2 In an effort to understand the role of ATG9A and ATG101 in suppressing inflammatory signaling, we found that loss of ATG9A and ATG101, but not ATG5 or ATG7, caused an accumulation of ubiquitin‐rich condensates. We also found that the accumulation of ubiquitin‐rich condensates coincided with an overactivation of the ubiquitin‐sensing, IRF3‐targeted kinase TBK1. Importantly, we found that inhibiting the accumulation of ubiquitin‐rich condensates via knock‐out of p62/SQSTM1 abrogated the increased inflammatory signaling caused by loss of ATG9A. Together, our data suggest a model in which the loss of basal autophagy machinery causes an accumulation of ubiquitin‐rich condensates, which, in turn, act as a platform for the aberrant activation of TBK1‐mediated inflammatory signaling. We propose that these data have important implications for diseases of protein aggregation wherein similar ubiquitin‐rich condensates form and aberrant inflammatory signaling plays a pathological role.References(1) Saitoh, T.; Fujita, N.; Hayashi, T.; Takahara, K.; Satoh, T.; Lee, H.; Matsunaga, K.; Kageyama, S.; Omori, H.; Noda, T.; Yamamoto, N.; Kawai, T.; Ishii, K.; Takeuchi, O.; Yoshimori, T.; Akira, S. Atg9a Controls DsDNA‐Driven Dynamic Translocation of STING and the Innate Immune Response. PNAS 2009, 106 (49). https://doi.org/10.1073/pnas.0911267106.(2) Kannangara, A. R.; Poole, D. M.; McEwan, C. M.; Youngs, J. C.; Weerasekara, V. K.; Thornock, A. M.; Lazaro, M. T.; Balasooriya, E. R.; Oh, L. M.; Soderblom, E. J.; Lee, J. J.; Simmons, D. L.; Andersen, J. L. BioID Reveals an ATG9A Interaction with ATG13‐ATG101 in the Degradation of P62/SQSTM1‐ubiquitin Clusters. EMBO reports 2021, 22 (10). https://doi.org/10.15252/embr.202051136.